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Gene Mosaicism Screening Using Single-Molecule Molecular Inversion Probes in Routine Diagnostics for Systemic
Benjamin Kant1, Ellen C Carbo1, Iris Kokmeijer1
1Department of Genetics, University Medical Center Utrecht, Utrecht, the Netherlands.
Abstract:
Diagnosis of systemic autoinflammatory diseases (SAIDs) is often difficult to achieve and can delay the start of proper treatments and result in irreversible organ damage. In several patients with dominantly inherited SAID, postzygotic mutations have been detected as the disease-causing gene defects. Mutations with allele frequencies <5% have been detected, even in patients with severe phenotypes. Next-generation sequencing techniques are currently used to detect mutations in SAID-associated genes. However, even if the genomic region is highly covered, this approach is usually not able to distinguish low-grade postzygotic variants from background noise. We, therefore, developed a sensitive deep sequencing assay for mosaicism detection in SAID-associated genes using single-molecule molecular inversion probes. Our results show the accurate detection of postzygotic variants with allele frequencies as low as 1%. The probability of calling mutations with allele frequencies ≥3% exceeds 99.9%. To date, we have detected three patients with mosaicism, two carrying likely pathogenic NLRP3 variants and one carrying a likely pathogenic TNFRSF1A variant with an allele frequency of 1.3%, confirming the relevance of the technology. The assay shown herein is a flexible, robust, fast, cost-effective, and highly reliable method for mosaicism detection; therefore, it is well suited for routine diagnostics.
Insights
Diagnosing systemic autoinflammatory diseases (SAIDs) is challenging. A new deep sequencing assay accurately detects low-frequency mosaic mutations, improving diagnosis and treatment for SAID patients.
Area of Science:
- Genetics
- Immunology
- Molecular Biology
Background:
- Systemic autoinflammatory diseases (SAIDs) pose diagnostic challenges, often leading to delayed treatment and organ damage.
- Postzygotic mutations, even at low allele frequencies, are implicated in some dominantly inherited SAIDs.
- Current next-generation sequencing methods struggle to differentiate low-grade mosaic variants from background noise.
Purpose of the Study:
- To develop a sensitive deep sequencing assay for accurate mosaicism detection in SAID-associated genes.
- To overcome the limitations of standard sequencing in identifying low-allele-frequency postzygotic mutations.
Main Methods:
- Development of a sensitive deep sequencing assay utilizing single-molecule molecular inversion probes.
- Application of the assay for mosaicism detection in genes associated with SAIDs.
Main Results:
- The assay accurately detects postzygotic variants with allele frequencies as low as 1%.
- The probability of correctly identifying mutations with allele frequencies ≥3% exceeds 99.9%.
- Three patients with mosaicism were identified, including two with likely pathogenic NLRP3 variants and one with a likely pathogenic TNFRSF1A variant (1.3% allele frequency).
Conclusions:
- The developed assay is a flexible, robust, fast, cost-effective, and reliable method for mosaicism detection in SAID-associated genes.
- This technology is well-suited for routine diagnostics, potentially improving early diagnosis and management of SAIDs.
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